Single family case studies
The studies of this case obtained the informed consent from all subjects. For publication of photos, consent was obtained from the patients.
The studies with animals follow the guidelines and ethical regulations. The research program and study protocols were approved by Animal Care and Ethical Committee of Nanjing Medical University (approval number 14030111).
Isolation of mouse articular chondrocytes and culture
Primary chondrocytes were isolated from 5–6 days old mice as described. Briefly, cartilages from tibial plateaus and femoral condyles were excised and all extraneous soft tissues were removed. To isolate chondrocytes, the cleaned cartilages were digested two consecutive times in cell culture medium with 3 mg/ml collagenase D (Roche, Indianapolis) at 37 °C for 45 min, and then overnight with 0.5 mg/ml collagenase D. The next day, the dislodged cells were passed through a 2 ml Pasteur pipet successively to disperse aggregates, then through a sterile 48-µm mesh before collected by centrifugation for 10 min at 400 x g, 20 °C. The cells were plated out at a density of 25 × 103/cm2. Chondrogenic differentiation was induced by replacing culture medium with DMEM/F12 (1:1), supplemented with 10% FBS, 1% pen&strep, 10 µg/ml insulin, 10 µg/ml transferrin, and 3 × 10 − 8 M sodium selenite. The differentiation medium was changed every three days.
Plasmids construction and cell transfection
The DNA fragment containing full length Dnm3os sequence was amplified by PCR from mouse genomic DNA and inserted between the BamHI and SalI sites in the pRK5 vector to generate pRK5-Dnm3os. PCR-based deletion strategy was used to generate the miR-199a-5p and miR-214 deletion mutant (DKO). The PCR primers used are as follows.
Primary articular chondrocytes were transfected with endotoxin-free plasmid constructs using Lipofectamine (Invitrogen) according to the manufacturer’s procedure.
Blood RNA extraction and RT-qPCR
10 ml blood from each subject was drawn with a BD Vacutainer CPT Cell Preparation Tube containing sodium citrate. Lymphocytes and monocytes were separated from the plasma in Ficoll solution. Briefly, the blood samples were diluted with 1:1 sterile PBS, and then carefully poured onto 10 ml Ficoll solution in a 50 ml centrifuge tube (the blood must remain on top, do not mix). The tubes were centrifuged for 20 min at 350xg, and lymphocytes and monocytes between plasma and Ficoll layers were harvested using a sterile pipette. The cells were washed twice with PBS, and the RNA was extracted using the RNAiso reagent to template cDNA synthesis using the PrimeScript RT reagent kit (TAKARA). SYBR green real-time qPCR reactions were carried out on a ABI7500 Real-Time PCR system. The cycling condition was 95 °C for 5 minutes, followed by 40 amplification cycles of 95 °C, 15 second and 60 °C, 1 minute. For each data point, triplicate reactions were carried out and the experiment was repeated three times to assess the statistical significance. RT-qPCR primer sequences are listed as follows.
PCR primers for human genes
homo BRAF-F: CCTCATTACCTGGCTCAC
PCR primers for mouse genes
Immunofluorescence staining and RNA-FISH
Cultured cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, and blocked for 30 min in PBS, 3%BSA, and 0.3% Triton X-100 prior to overnight incubation with primary antibodies. Anti-Sox9 and anti-Col2a1 (abcam) were used at 1:1000. Cells grown on cover slips for RNA-FISH were fixed in 3.7% formaldehyde solution at room temperature for 10 minutes, then permeabilized with 70% cold ethanol for at least an hour. Stellaris probe was added in 100 µL of hybridization buffer (Biosearch, Inc.) and incubateed in a dark humidified chamber at 37 °C for 4 hours. The cells were visualized with DAPI counterstaining (5 ng/mL) under a wide field fluorescence microscope.
Edu incorporation assay
To measure cell growth, 24 hours after transfection in 24-well plates, 20 mM EdU was added for 8 hour. The cells were then fixed in 3.7% formaldehyde, then washed with PBS and permeablized. 500 µl Click-iT reaction cocktail (430 ml 1xClick-iT reaction buffer, 20 ml CuSO4, 1.2 ml Alexa Fluor® azide, and 50 ml reaction buffer additive) was added to each well and incubated for 45 min at the room temperature in the dark. The cells were counter-stained with DAPI for nuclei and visualized under an inverted fluorescence microscope. Images were processed with Image J and the percentage of EdU incorporation was calculated based on the number of EdU positive (red) and total (DAPI) cells.
Alcian blue staining and quantification
Chondrocytes were cultured for 14 days in chondrogenic differentiation medium, then fixed in 4% formalin for 10 min. After washing twice with PBS, the cells were incubated with 3% acetic acid for 10 min and stained with 1% alcian blue in 3% acetic acid (pH 2.5) for 30 min and photographed. For quantification, the stained cells were washed twice, and the alcian blue dye was extracted with 500 ml dimethyl sulfoxide. Absorbance was measured at 650 nm.
Alkaline phosphatase assay
Histochemical detection of alkaline phosphatase activity was performed on cells that were fixed for 2 min in 4% paraformaldehyde at room temperature. After washing with TBST, the cells were incubated for 30 min in in 0.1 M Tris-HCl, pH 8.5, containing 0.1 mg/ml Naphthol AS-MX phosphate, 0.5% N,N-dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml fast blue BB salt, and then photographed.
In vitro differentiation of chondrogenic ATDC5 cells
ATDC5 cell line was culture in DMEM/F-12 medium supplemented with 5% FBS, penicillin (100units/mL)/streptomycin (0.1 mg/mL), and 4 mM L-Glutamine. For differentiation experiments, ATDC5 cells were seeded at 80–90% confluence in 12-well plate. After reaching 100% confluence, ATDC5 cells were incubated with serum-free medium for 24 hours, then exposed to differentiating medium containing 1% Insulin-transferrin-sodium selenite (ITS, Sigma-Aldrich) and 50 nM Vitamin C.
RNA Sequencing and Data Analysis
RNA from Rk5-vector, Full length and DKO mutant Dnm3os transfected primary mouse articular chondrocytes were extracted using TRIzol (Life Technol- ogies), followed by purification using a RNeasy Mini Kit (Qiagen). RNA-seq was performed using primary mouse articular chondrocytes from two individual animals. RNA-seq libraries were prepared using the Illumina TruSeq RNA Library Prep Kit v2 and sequenced by a HiSeq 2500 sequencer. RNA-seq reads were aligned to mm10 using TopHat with default settings (http://tophat. cbcb.umd.edu/).
Prism 8.0 (GraphPad software) was employed for analyses. Data from a minimum of three independent experiments were presented as mean ± s.d. Animals in the experiment were randomly selected and grouped. An unpaired two-tailed Student’s t-test was used for analyzing two data sets, and one-way analysis of variance was used for more sets. The significance threshold was set at 0.05 (p < 0.05).